The HIV-1 nucleocapsid protein (NC) is a nucleic acid chaperone, which catalyzes the rearrangement of nucleic acids into what is presumably their most thermodynamically stable structure. Most retroviruses use a host cell transfer RNA (tRNA) to prime minus-strand DNA synthesis. Human tRNA (Lys,3) is selected as the natural primer for HIV-1 reverse transcriptase (RT). Among its numerous chaperone functions, NC has been shown to facilitate the annealing of the 18 nucleotides at the 3? end of tRNA (Lys,3) to the complementary primer binding site (PBS) on the HIV RNA genome. This tRNA annealing step is the first step in a complex series of events that ultimately result in the synthesis of double-stranded proviral DNA. This proposal is based on the observation that specific molecular interactions occur between the tRNA (Lys,3) primer, the HIV RNA genome, and NC. The details of these interactions and the mechanism of NC?s nucleic acid chaperone function, however, are not well understood. The complexes formed by human tRNA (Lys,3), the RNA genome, NC, and other intermediates in retroviral reverse transcription are attractive targets for new therapies. Therefore, we propose: A) To probe the mechanism of HIV- 1 NC-mediated tRNA primer/RNA template initiation complex formation by terbium cleavage assays, in vitro selection (SELEX), nucleotide analog interference mapping (NAIM) experiments, and kinetic assays, B) To probe the mechanism of minus-strand transfer mediated by HIV-1 NC using fluorescence resonance energy transfer (FRET), and C) To probe the mechanism of HIV-1 NC?s nucleic acid chaperone activity by single molecule DNA and RNA stretching using an optical tweezers apparatus.